U.S. patent application number 11/616483 was filed with the patent office on 2007-07-19 for method for manufacturing protein chip substrate using plasma and protein chip substrate manufactured by the method.
This patent application is currently assigned to Sungkyunkwan Univ. Foundation For Corporate Coll.. Invention is credited to Chang-rok Choi, Dong-geun Jung, Sang-hak Yeo.
Application Number | 20070166815 11/616483 |
Document ID | / |
Family ID | 38263673 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166815 |
Kind Code |
A1 |
Jung; Dong-geun ; et
al. |
July 19, 2007 |
METHOD FOR MANUFACTURING PROTEIN CHIP SUBSTRATE USING PLASMA AND
PROTEIN CHIP SUBSTRATE MANUFACTURED BY THE METHOD
Abstract
Disclosed herein are a protein chip substrate and a method for
manufacturing the protein chip substrate. The method includes
deposition of plasma polymerized ethylenediamine (PPEDA) having an
amine group on plasma polymerized cyclohexnane (PPCHex) by
inductively coupled plasma-chemical vapor deposition (ICP-CVD),
thereby preventing non-specific adsorption of proteins on a slide
surface.
Inventors: |
Jung; Dong-geun;
(Gangnam-gu, KR) ; Yeo; Sang-hak; (Gangbuk-gu,
KR) ; Choi; Chang-rok; (Gunpo-si, KR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Sungkyunkwan Univ. Foundation For
Corporate Coll.
Suwon-si
KR
|
Family ID: |
38263673 |
Appl. No.: |
11/616483 |
Filed: |
December 27, 2006 |
Current U.S.
Class: |
435/287.2 ;
427/2.11; 977/902 |
Current CPC
Class: |
C07K 1/1077
20130101 |
Class at
Publication: |
435/287.2 ;
427/002.11; 977/902 |
International
Class: |
C12M 3/00 20060101
C12M003/00; B05D 3/00 20060101 B05D003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2005 |
KR |
10-2005-0131921 |
Claims
1. A method for manufacturing protein chip substrate wherein plasma
polymerized ethylenediamine (PPEDA) having an amine group is
deposited on plasma polymerized cyclohexnane (PPCHex) by
inductively coupled plasma-chemical vapor deposition (ICP-CVD).
2. The method according to claim 1, the method comprising the steps
of: (a) depositing a plasma-polymerized cyclohexane (PPCHex) film
using cyclohexane as a precursor on a slide glass as a substrate by
inductively coupled plasma-chemical vapor deposition (ICP-CVD); and
(b) depositing a plasma-polymerized ethylenediamine (PPEDA) film
using ethylenediamine (EDA) as a precursor on the PPCHex film by
inductively coupled plasma-chemical vapor deposition (ICP-CVD).
3. The method according to claim 2, wherein the plasma-polymerized
cyclohexane (PPCHex) film is maintained at a substrate temperature
of 27.degree. C.
4. The method according to claim 2, wherein the plasma-polymerized
cyclohexane (PPCHex) film is deposited using a carrier gas selected
from Ar, N.sub.2, He and H.sub.2.
5. The method according to claim 4, wherein, during step (a), the
carrier gas is used at a flow ratio of about 20 sccm.
6. The method according to claim 5, wherein, during step (a), the
deposition is conducted under a plasma reaction chamber inner
pressure of about 300 mtorr for 30 sec.
7. The method according to claim 4, wherein, during step (a), the
deposition is conducted under a plasma reaction chamber inner
pressure of about 300 mtorr for 30 sec.
8. The method according to claim 4, wherein, during step (b), the
carrier gas is used at a flow ratio of about 15 sccm.
9. The method according to claim 8, wherein, during step (b), the
deposition is conducted under a plasma reaction chamber inner
pressure of about 30 mtorr for 2 min.
10. The method according to claim 4, wherein, during step (b), the
deposition is conducted under a plasma reaction chamber inner
pressure of about 30 mtorr for 2 min.
11. The method according to claim 2, wherein, during step (a), ICP
power applied from a RF generator of the plasma reaction chamber to
an outer electrode is maintained at about 15 W, and SB power
applied from the RF generator to a substrate supporter as an inner
electrode is maintained at 10 W to 70 W.
12. The method according to claim 2, wherein, during step (b), ICP
power applied from a RF generator of the plasma reaction chamber to
an outer electrode is maintained at about 4 W, and SB power applied
from the RF generator to a substrate supporter as an inner
electrode is maintained at about 4 W.
13. A method for manufacturing a protein chip substrate, the method
comprising the steps of: washing a slide glass with
trichloroethylene, acetone and methanol, sequentially; arranging
the slide glass on a substrate supporter inside a plasma reaction
chamber; controlling an inner pressure of the plasma reaction
chamber by a vacuum device; feeding cyclohexane as a precursor
together with a carrier gas into the plasma reaction chamber by a
bubbler; controlling an inner temperature of the plasma reaction
chamber by a heater under the substrate supporter; applying
inductively coupled plasma (ICP) power and slide voltage (SB) power
to an outer electrode and the substrate supporter by the RF
generator, respectively, to generate plasma between the slide glass
and the plasma reaction chamber; uniformly depositing a plasma
polymersized cyclohexane (PPCHex) film on the slide glass while
polymerizing cyclohexane as a precursor by plasma; and depositing a
plasma-polymerized ethylenediamine (PPEDA) film using
ethylenediamine (EDA) as a precursor on the PPCHex film by
inductively coupled plasma-chemical vapor deposition (ICP-CVD).
14. A protein chip substrate manufactured by the method according
to claim 1.
15. A protein chip substrate manufactured by the method according
to claim 2.
16. A protein chip substrate manufactured by the method according
to claim 13.
17. A protein chip substrate comprising a plasma polymerized
cyclohexnane (PPCHex) film deposited on a substrate by inductively
coupled plasma-chemical vapor deposition (ICP-CVD), and a plasma
polymerized ethylenediamine (PPEDA) film having an amine group
deposited on the PPCHex film.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a protein chip substrate
and a method for manufacturing a protein chip substrate. More
specifically, the present invention relates to a method for
manufacturing a protein chip substrate wherein plasma polymerized
ethylenediamine (PPEDA) having an amine group is deposited on
plasma polymerized cyclohexnane (PPCHex) being free of non-specific
adsorption by inductively coupled plasma-chemical vapor deposition
(ICP-CVD), thereby preventing non-specific adsorption of proteins
on a slide surface, and a protein chip substrate manufactured by
the method.
[0003] 2. Description of the Related Art
[0004] With recent discoveries of human genomic structures,
research for human genomic functions has increasingly attracted
considerable attention.
[0005] There have been advances in a new research field referred to
as "functional proteomics" for systematically probing functions of
human genes.
[0006] Functional proteomics is divided into three groups of
"genomics", "proteomics" and "bioinformatics". Genomics is focused
on studies targeting genes, proteomics is focused on studying the
behavior of genes by targeting overall proteins in cells, and
bioinformatics is a combined-approach of the two fields.
[0007] Molecular biological access to function studies for genes or
cells was made on the basis of controlling a single gene or mRNA
expressed by the gene. Nowadays, the recent trend towards
proteome-based analysis is being introduced.
[0008] Protein chips are required for the proteome-based analysis
as a key technique (Gavin MacBeath and Stuart L. Schreiber,
Science, 289:1760.about.1763, 2000).
[0009] The protein chips are an automatic system capable of
simultaneously analyzing binding structure of a large amount of
proteins by immobilization of tens to thousands of proteins on a
small substrate.
[0010] The protein chips are similar to microarray DNA chips which
detect gene expressions or mutants by immobilizing tens to
thousands of genes on a small substrate, and simultaneously
analyzing variations in a large amount of genes through the
behavior of complementary bindings between genes (Yasuda, H.,
Lamage, C. E., J. A ppl. Polym. Sci.17: 201, 1973; Muguruma, H.,
Karube, I., Trends Anal. Chem. 18: 62, 1999; Nalanishi, K.,
Muguruma, H., Karube, I., Anal. Chem. 68: 1695, 1996; Miyachi, H.,
Hiratsuka, A., Ikebukuro, K., Yano, K., Muguruma, H., Karube, I.,
Biotechnol. Bioengin.69: 323, 2000).
[0011] However, protein chips can be used for the following
analysis unattainable by DNA chips.
[0012] First, they can be used to obtain information regarding
interaction between protein-protein. The signal transmission or
control within cells is represented as the interaction between
protein-protein. Thus, this can be analyzed by protein chips.
[0013] Second, proteins, from higher organisms including humans to
yeasts, undergo post-translational modification. As a result, it is
possible to obtain information regarding a secondary modification
such as phosphorylation and oxidation.
[0014] Third, proteins enable detection of problems which may be
generated after mRNA formation. A variety of diseases are caused by
problems associated with post-transcription control, protein
generation and protein localization. DNA chips used to detect mRNAs
make it difficult to obtain information regarding the problems.
[0015] Fourth, when proteins are expressed from mRNAs, correlation
in quantity between the proteins and mRNAs is often not
sufficiently high. Thus, in some cases, DNA chips have limitation
of difficulty in obtaining correct information associated with
proteins. However, protein chips enable solving such limitation of
DNA chips.
[0016] Fifth, difference in base sequence of genes revealed by DNA
chips is not directly indicative of disease or difference in
phenotype. Furthermore, since mutants derived from amino acids
having similar characteristics generally retain inherent
characteristics of proteins, they hardly cause disease or
difference in phenotype.
[0017] On the other hand, the use of protein chips enables
identification of the protein inducing disease or difference in
phenotype.
[0018] In addition, the use of protein chips ensures database
formation for relationship between protein-protein, and is
effective in reducing time and costs upon development of novel
medicines. For example, a period required to develop a treatment
medicine using protein chips can be considerably shortened, as
compared to the cases using current genes.
[0019] Protein chips are utilized in a wide variety of industrial
applications including disease diagnosis, environment monitoring
and harmfulness testing, as well as industrial applications.
[0020] Based on the mentioned advantages, protein chips are
expected to have high marketability, as compared to DNA chips.
Thus, there is an eager demand for developing protein chips.
[0021] Such a protein chip includes a sensor chip and a protein
detection system.
[0022] The sensor chip is a major constituent for determining a
developing rate of the protein chip.
[0023] The sensor chip has a structure in which tens to thousands
of proteins are arranged in a predetermined array on the surface of
a small chip. The chip is selected from a slide-sized glass or
plastic.
[0024] A key technique associated with the sensor chip is protein
immobilization for attaching proteins to the surface thereof.
[0025] The protein immobilization techniques are classified into
three groups, based on the characteristics.
[0026] First, immobilization of a specific protein to the surface
of sensor chips using carboxymethyl-dextran (CM) is the most widely
used method. Here, amine coupling is the most commonly used.
Alternately, thiol coupling or avidin-biotin coupling is used to
immobilize acid proteins or DNAs.
[0027] Second, surface treatment of sensor chips is suitable for
immobilizing a plurality of protein groups having the same
characteristics.
[0028] Third, poly lysine or calyx crown is available for
immobilizing large amount of non-specific proteins.
[0029] The control of protein immobilization and patterned array of
biomolecules are implicated in wide a variety of fields including
basic studies of biochips, bioelectronics and cell-biology.
[0030] A variety of techniques, e.g., self-assembled monolayer and
lithography, are generally used for protein immobilization.
[0031] In recent years, studies for immobilizing bio molecules on a
solid slide using plasma polymerized films have been actively
investigated.
[0032] Plasma is a state in which neutral gas molecules absorb
electrical or thermal energy, and are then separated into ions and
electrons.
[0033] Studies associated with techniques utilizing plasma have
been actively made.
[0034] Plasma is being gradually expanded to a variety of
applications including plasma etching and plasma enhanced chemical
vapor deposition (PECVD) in semiconductor device fabrication,
surface treatment of metals or polymers, synthesis of enhanced
materials (e.g., imitation diamonds), plasma display panels (PDPs)
and environment protection techniques.
[0035] Plasma partially ionizes gas, and efficiently activates
molecules.
[0036] Relatively inert molecules are exposed to plasma, thereby
being readily activated.
[0037] Coating of plasma polymerized films is achieved by arranging
monomers in a deposition chamber, activating or decomposing
reactants with plasma, and allowing the reactants to be condensed
on the surface of a slide, to form a polymerized film.
[0038] When precursors containing an amine or aldehyde group are
used, plasma polymerized films can contain a large amount of the
group. Thus, such solid slide is utilized in biomolecules
immobilization.
[0039] The plasma polymerized films are largely different with
chemically-polymerized general films.
[0040] Such plasma polymerized film has no porosity, is
mechanically and chemically stable, and has an adhesive property to
the slide due to its cross-linked structure.
[0041] In addition, the plasma polymerized film has superior
controllability in thickness and high uniformity, as compared to
chemically-polymerized films. These advantages provide suitability
for manufacture of protein and DNA arrays.
[0042] To achieve highly sensitive diagnostic protein chips, a
sufficient amount of proteins must be immobilized at a
predetermined space thereof. Accordingly, there is a demand for
methods that can immobilize proteins more efficiently.
[0043] Protein immobilization based on covalent bonds depends on
nucleophilicity or electrophilicity due to chemical or physical
formation on the surface of the slide glass.
[0044] A well-known problem in experiments associated with protein
chips is non-specific adsorption of proteins on the surface of the
slide glass. The non-specific adsorption is misrecognized as
obviously false response in diagnostic inspection.
[0045] However, conventional protein chips have a problem of
occurrence of non-specific adsorption even in a region where no
amine group exists.
[0046] Thus, there is a need for techniques that minimize
non-specific adsorption upon diagnosis based on protein chips.
SUMMARY OF THE INVENTION
[0047] It is an object of the present invention to prevent
non-specific adsorption of proteins on a slide surface in protein
immobilization.
[0048] To accomplish the above-mentioned object, the present
invention suggests that plasma polymerized ethylenediamine
(hereinafter, referred to simply as "PPEDA") having an amine group
is deposited on plasma polymerized cyclohexnane (hereinafter,
referred to simply as "PPCHex") being free of non-specific
adsorption by inductively coupled plasma-chemical vapor deposition
(ICP-CVD).
[0049] Ethylenediamine and cyclohexnane are used as precursors for
the PPEDA and PPCHex, respectively.
[0050] Detailed modification of chemical properties of a slide
glass is required for protein immobilization.
[0051] The PPCHex was deposited on the slide glass. As a result,
the surface of the slide glass becomes hydrophilic.
[0052] The PPEDA was patterned on the resulting structure
(PPCHex/glass slide) using a patterned mask.
[0053] The hydrophilicity or hydrophobicity of each film is
determined by measuring the contact angle between water and the
film.
[0054] The contact angle between water and the surface of PPEDA
film was about 45.degree.. This indicates that the PPEDA film
surface is hydrophilic.
[0055] On the other hand, the contact angle between water and the
surface of PPCHex film was equal to or greater than 90.degree..
This indicates that the PPCHex film surface is hydrophobic.
[0056] The PPEDA film deposited under suitable conditions hardly
had non-specific binding.
[0057] The PPCHex film being free of non-specific adsorption was
evaluated for roughness and chemical structure with an atomic force
microscopy (APM) and Fourier transform infrared spectroscopy,
respectively.
[0058] Immobilized proteins and the degree of non-specific
adsorption are measured by fluorescence detection with an exciting
wavelength of 490 nm and an emitting wavelength of 520 nm.
[0059] The immobilized proteins according to the present invention
are observed only on the hydrophilic PPEDA patterned on PPCHex film
being free of non-specific adsorption.
[0060] As a result, the inventors found that proteins tend to be
immobilized on the PPEDA being hydrophilic and having an amine
group, exclusively.
[0061] The method of the present invention enables proteins to be
immobilized in a desired region and proteins to be arranged in a
predetermined position, thus being very valuable.
[0062] Accordingly, patterning techniques of plasma polymerized
cyclohexnane (PPCHex) being free of non-specific adsorption on
plasma polymerized ethylenediamine (PPEDA) having an amine group
suggests an advanced technical trend in biochip analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The above and other objects, features and other advantages
of the present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0064] FIG. 1 is a diagram schematically illustrating an
inductively coupled plasma-chemical vapor deposition (ICP-CVD)
system used in manufacture of a protein chip substrate according to
the present invention;
[0065] FIG. 2 are photographs illustrating the static contact angle
between water and each film measured by a charge-coupled device
(CCD), respectively;
[0066] FIG. 3 are photographs illustrating non-specific protein
immobilization on each PPCHex slide glass;
[0067] FIG. 4A are photographs illustrating an average
signal-to-noise ratio and fluorescence intensity of each PPEDA
slide glass before imaging;
[0068] FIG. 4B are photographs illustrating an average
signal-to-noise ratio and fluorescence intensity of each PPEDA
slide glass after imaging;
[0069] FIG. 4C is a graph illustrating changes in average
signal-to-noise ratio of a PPEDA slide glass as an increasing
function of plasma power;
[0070] FIG. 5A is a graph illustrating a FTIR spectrum of a PPCHex
film;
[0071] FIG. 5B is a graph illustrating changes in peak of a
hydroxyl group (--OH) as a function of deposition power; and
[0072] FIG. 6 are photographs illustrating a surface roughness of
each PPCHex film.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0073] Detailed description will be made of preferred embodiments
of the present invention with reference to the accompanying
drawings. However, various changes and modifications may be made to
the preferred embodiments of the invention and the invention is not
to be construed as being limited to the embodiments.
[0074] According to a method for manufacturing a protein chip of
the present invention, attaching precursor molecules to a substrate
is preferably carried out by inductively coupled plasma-chemical
vapor deposition (ICP-CVD).
[0075] FIG. 1 is a diagram schematically illustrating an ICP-CVD
system used in manufacture of a protein chip substrate according to
the present invention.
[0076] The ICP-CVD system includes a plasma reaction chamber 1, a
vacuum device 7 to control a pressure inside the plasma reaction
chamber 1, a bubbler 9 to allow a precursor 11 in the form of gas
to be fed into the plasma reaction chamber 1, a shower ring 10 to
spray the vaporized precursor from the bubbler 9 to the plasma
reaction chamber 1, an outer electrode 2 arranged on the plasma
reaction chamber 1, a substrate supporter 4 to support a substrate
3 and function as an inner electrode, the substrate supporter 4
arranged inside the plasma reaction chamber 1, a radio frequency
generator (RF generator) 6 to supply inductively coupled plasma
(ICP) power to the outer electrode 2 and supply slide voltage (SB)
power to the substrate supporter 4.
[0077] The ICP-CVD system further includes a gas repository to
store a carrier gas such as He and Ar (not shown), a flow regulator
to control moles of passing gas (not shown), a cooler to control a
temperature inside the chamber (not shown) and a fan to promote
convection inside the chamber (not shown).
[0078] The substrate supporter 4 is provided with a heater
therein.
[0079] A method for depositing a thin film using the ICP-CVD system
to manufacture a protein chip substrate according to the present
invention will be described as follows.
[0080] First, a plasma-polymerized cyclohexane (PPCHex) film using
cyclohexane (CHex) as a precursor was deposited on a slide glass as
a substrate by ICP-CVD.
[0081] Next, a plasma-polymerized ethylenediamine (PPEDA) film
using ethylenediamine (EDA) as a precursor was deposited on the
PPCHex film by ICP-CVD.
[0082] A method for depositing the PPCHex film on the substrate
will be described as follows.
[0083] The substrate 3 was sequentially washed with
trichloroethylene, acetone and methanol, and was then arranged on
the substrate supporter 4 inside the plasma reaction chamber 1.
[0084] Next, the pressure inside the chamber 1 was controlled by
the vacuum device 7. Cyclohexane as a precursor together with the
carrier gas was fed into the chamber 1 by the bubbler 9. The
temperature inside the chamber 1 was elevated by the heater under
the substrate supporter 4.
[0085] When inductively coupled plasma (ICP) power and slide
voltage (SB) power are applied to the outer electrode 2 and the
substrate supporter 4 by the RF generator 6, respectively, plasma
generated by the outer electrode 2 and the substrate supporter 4
(inner electrode) was formed between the substrate 3 and the
chamber 1.
[0086] At this time, cyclohexane was polymerized by the plasma, the
plasma polymerized cyclohexane (PPCHex) film was uniformly
deposited on the substrate 3.
[0087] The deposition chamber used in the present invention has a
structure of cylinder and is made of a stainless material. The
chamber has a diameter of 30 cm and a height of 28 cm.
[0088] Cyclohexane monomer was put in the bubbler 9 adjusted to a
temperature of 50.degree. C.
[0089] Cyclohexane was vaporized by using Ar as inert carrier gas,
and was then fed into the chamber 1.
[0090] In addition, SB power generates plasma around the slide
owing to a high-frequency generator arranged on the substrate
supporter 4.
[0091] The walls of the deposition chamber 1 were grounded.
[0092] The deposition chamber 1 was maintained at a base pressure
of approximately 10.sup.-5 Torr, by vacuum device 7.
[0093] The PPEDA film was maintained at a temperature of 27.degree.
C.
[0094] The precursors were preferably fed into the chamber 1 by
using a carrier gas. The examples of the carrier gas include Ar,
N.sub.2, He and H.sub.2. More preferred is Ar.
[0095] The PPEDA film was deposited on the slide glass used as the
substrate 3, while maintaining Ar flow ratio of about 20 sccm.
[0096] The deposition was carried out under a pressure of 300 mtorr
for 30 sec.
[0097] The ICP power applied from the RF generator 6 to the outer
electrode was preferably maintained at 15 W.
[0098] The SB power applied from the RF generator 6 to the
substrate supporter 4 as an inner electrode was elevated from 10 W
to 70 W.
[0099] Subsequently, a plasma-polymerized ethylenediamine (PPEDA)
film using ethylenediamine (EDA) as a precursor by ICP-CVD was
deposited on the PPCHex film through a patterned mask, thereby
patterning the PPEDA film.
[0100] At this time, the deposition was conducted at room
temperature and an Ar flow of about 30 sccm. The deposition was
conducted at a pressure of 30 mtorr for about 2 min. The ICP and SB
powers were maintained at 4 W and 3 W, respectively.
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
and fluorescein isothiocyanate (FITC)-conjugated Goat anti-Rabbit
immunoglobulin G (IgG) were used in Examples of the present
invention. In addition, a diluted solution of each reagent in
phosphate buffered saline (PBS) was used in Examples.
[0101] As plasma deposition system used in Examples of the present
invention, any outer electrode and substrate supporter may be used
without any particular limitation in terms of material and
structure so long as they are commonly used in chemical vapor
deposition systems.
[0102] In particular, the outer electrode is preferably formed of a
flat circular coil. The outer electrode is preferably made of
material being chemically stable and being in little danger of
contamination. The more preferred material is a stainless
material.
[0103] The substrate is preferably made of one selected from the
group consisting of glass, plastic, metal and silicone. Preferred
is glass.
[0104] Efficiency of protein immobilization in films manufactured
according to the present invention was evaluated by measuring
contact angle, fluorescence, surface roughness by atomic force
microscope (AFM), and chemical structure by Fourier transform
infrared spectroscopy (FTIR) The evaluation will be described in
detail as follows.
[0105] Contact Angle Measurement
[0106] The present inventors measured the static contact angle
between water of 2 .mu.l and samples. The measurement was based on
the digital image recoded with a charge-coupled device (CCD) as
equal to the level of the sample.
[0107] The static contact angle measurement enables evaluation of
the degree of hydrophilicity or hydrophobicity.
[0108] The results are shown in FIG. 2.
[0109] The contact angle between water and the surface of PPEDA
film was about 45.degree.. This indicates that the PPEDA film
surface is hydrophilic (FIG. 2F).
[0110] An amine group is reported to have hydrophilicity and a
superior protein-binding capability.
[0111] On the other hand, the contact angle between water and the
surface of PPCHex film was equal to or greater than 90.degree..
This indicates that the PPCHex film surface is hydrophobic (FIGS.
2A to 2E).
[0112] Hydrophobic surfaces mean that a weak interaction exists
between water and the film. The film surfaces have a hydrocarbon
group (--CHx) only.
[0113] Meanwhile, hydrophilic surfaces mean that a strong
interaction exists between water and the film. The film surfaces
have an oxygen group.
[0114] Protein Immobilization
[0115] Patterned slide glasses were cultured in
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
solution at room temperature for 10 min. Then, the slide glasses
were washed with deionized water.
[0116] 4 ng/uL of fluorescein isothiocyanate (FITC)-conjugated Goat
anti-Rabbit immunoglobulin G (IgG) solution was cultured on the
slide glasses for 1 hour.
[0117] The resulting IgG culture was washed with deionized water,
followed by drying.
[0118] Fluorescence Measurement
[0119] The resulting protein-immobilized FITC-conjugated IgG was
scanned with a laser fluorescent scanner.
[0120] The protein-immobilized FITC-conjugated IgG was excited at
490 nm and emitted at about 520 nm.
[0121] Non-specific protein adsorption on each of the PPCHex slide
glasses was analyzed for fluorescence measurement.
[0122] The PPCHex slide glasses deposited at different powers were
prepared. Each of the PPCHex slide glasses was evaluated for
suitability for use in protein chips.
[0123] FIGS. 3A to 3E are CCD fluorometric images confirming
non-specific protein immobilization on the PPCHex slide glasses,
respectively.
[0124] As can be seen from FIGS. 3A to 3E, as SB power is elevated
from 10 W to 70 W, non-specific protein adsorption decreases.
[0125] In particular, non-specific protein adsorption was not shown
at a plasma SB power of 70 W.
[0126] FIGS. 4A to 4C show an average signal-to-noise ratio and
fluorescence intensity of PPEDA slide glasses deposited on PPCHex
films and a PPEDA slide glass, respectively.
[0127] FIG. 4A shows an average signal-to-noise ratio and
fluorescence intensity of a PPEDA slide glass before imaging.
[0128] FIG. 4B shows an average signal-to-noise ratio and
fluorescence intensity of a PPEDA slide glass after imaging.
[0129] FIG. 4C is a graph showing comparison of changes in average
signal-to-noise ratio of a PPEDA slide glass as an increasing
function of plasma power.
[0130] In FIG. 4A, (a) shows an average signal-to-noise ratio of a
PPEDA slide glass deposited on a PPCHex film at a plasma SB power
of 10 W. (b) shows an average signal-to-noise ratio of a PPEDA
slide glass deposited on a PPCHex film at a plasma SB power of 30
W. (c) shows an average signal-to-noise ratio of a PPEDA slide
glass deposited on a PPCHex film at a plasma SB power of 50 W. (d)
shows an average signal-to-noise ratio of a PPEDA slide glass
deposited on a PPCHex film at a plasma SB power of 70 W. (e) shows
an average signal-to-noise ratio of a PPEDA slide glass deposited
on a bare glass substrate.
[0131] As a result, the inventors have confirmed that SB deposition
power has a great influence on the reduction in non-specific
protein adsorption.
[0132] As can be seen from FIGS. 4A to 4C, the average
signal-to-noise ratios for respective samples in accordance with
variant deposition power were 3.36, 3.96, 4.21 and 4.92. Meanwhile,
the average signal-to-noise ratio of the PPEDA slide glass
deposited on the bare glass substrate without any PPCHex film was
2.81.
[0133] In the PPEDA slide glass deposited on the bare glass
substrate without any PPCHex film, noise is increased, as signal is
increased. On the other hand, in the PPEDA/PPCHex film deposited at
a plasma SB power of 70 W, signal is small and noise hardly
occurs.
[0134] Chemical Structure and Composition Analysis by FTIR
[0135] To find the cause of non-spesific adsorption, chemical
structure and composition of the films were evaluated by Fourier
transform infrared spectroscopy (FTIR).
[0136] FTIR spectra are normalized to the same peak value.
[0137] FIG. 5A shows a FTIR spectrum of the PPCHex film.
[0138] FIG. 5B is a graph illustrating changes in peak of a
hydroxyl group (--OH) as a function of deposition power.
[0139] As shown in FIG. 5A, at SB powers of 10 W and 30 W, broad
signals appear at frequencies of 3400 cm.sup.-1 and 1650 cm.sup.-1.
This is the reason that moisture in air was adsorbed on the surface
of PPCHex film deposited at 10 W and 30 W.
[0140] High signal peaks at 3400 cm .sup.-1 and 1650 cm.sup.-1
indicate that the PPCHex film surface has a hydroxyl group (--OH)
which is generated from oxidation therein.
[0141] When a low plasma SB power of 10 W to 30 W is applied,
cyclohexane precursors are incompletely decomposed, and are
adsorbed in the form of a monomer.
[0142] At this time, the film is not densely packed. Accordingly,
the film is exposed to air at room temperature, moisture is
adsorbed on the surface thereof.
[0143] On the other hand, when a high plasma SB power of 50 W to 70
W is applied, cyclohexane precursors are completely decomposed, and
are adsorbed in the form of a diamond-carbon.
[0144] In the FTIR spectrum, there is no signal peak at 3400
cm.sup.-1 and 1650 cm.sup.-1. But, peak of hydrocarbon group
(--CHx, x=2 or 3) signal only appeared.
[0145] The PPCHex films deposited at different powers are compared.
As a result, it could be confirmed that the PPCHex films were
greatly different in chemical structure. This indicates that the
film structure depends on the deposition power.
[0146] There has been reported that a hydroxyl (--OH) or aldehyde
(--CHO) group present on the surface of glass induces chemical
protein adsorption.
[0147] However, as shown in FIG. 5B, a peak for hydroxyl (--OH) or
aldehyde (--CHO) hardly appears in the PPCHex film deposited at 70
W.
[0148] Relative area ratio of hydroxyl (--OH) peaks was decreased
as a plasma power was increased.
[0149] It has been already reported that the difference in
structure between the films considerably depends on the replacement
of hydrophobic methyl (--CH.sub.3) by hydrophilic hydroxyl
(--OH).
[0150] Existence of such hydroxyl is indicative of formation of
aldehyde, carbonyl and carboxyl, and thus occurrence of
non-specific adsorption.
[0151] The overall PPCHex film exhibited considerably greater
hydrophobicity. The PPCHex film deposited at 70 W has no adsorption
of moisture or oxygen therein owing to its high denseness.
[0152] Surface Roughness Measurement by AFM
[0153] The slide glass was evaluated for surface roughness with an
atomic force microscopy (AFM).
[0154] The AFM measurement was conducted in a contact mode and a
measurement range of 1 .mu.m.
[0155] FIG. 6 is a photograph illustrating a surface roughness of
the PPCHex film.
[0156] As shown in FIG. 6, root mean square (RMS) values of surface
roughness for samples at respective deposition powers were
approximately 0.08854 nm, 0.1457 nm, 0.2282 nm and 0.2611 nm.
[0157] The PPCHex film deposited at a high power was rougher than
the PPCHex films deposited at a low power. This is the reason that
the surface of films is exposed to plasma upon deposition of PPCHex
films. As a result, ionic or charged groups generated within the
films by plasma at increased plasma power damaged the film
surfaces.
[0158] The damage caused by such plasma is increased by elevating a
plasma exposure power (i.e., deposition power). As a result, the
PPCHex film surface was roughened.
[0159] It has already been reported in some cases that proteins
tend to be readily adsorbed on a relatively rough surface.
[0160] From the results, it could be estimated that root mean
square (RMS) values of the roughness were varied, but no physical
protein adsorption occurred.
[0161] This estimation is based on the fact that the PPCHex film
deposited at 70 W does not undergo considerable protein adsorption,
although it has the highest roughness.
[0162] Accordingly, protein adsorption is caused by chemical
adsorption, rather than physical adsorption.
[0163] As apparent from the above description, according to the
method of the present invention, plasma polymerized ethylenediamine
(PPEDA) having an amine group is deposited on plasma polymerized
cyclohexnane (PPCHex) being free of non-specific adsorption by
inductively coupled plasma-chemical vapor deposition (ICP-CVD).
[0164] Ethylenediamine and cyclohexnane are used as precursors for
the PPEDA and PPCHex, respectively.
[0165] The PPEDA patterned on the PPCHex was subjected to protein
immobilization.
[0166] The overall protein was immobilized on the hydrophilic
PPEDA, rather than the PPCHex.
[0167] The PPEDA/PPCHex film exhibits improvement in
signal-to-ratio value, as compared to the PPEDA film merely
patterned without depositing any PPCHex.
[0168] The surface of the PPEDA/PPCHex film undergoes changes in
chemical structure. The surface roughness is slightly increased
from 0.08854 nm to 0.2611 nm, as deposition power is elevated from
10 W to 70 W.
[0169] As can be seen from the results, protein adsorption on the
PPCHex film is caused by chemical adsorption, rather than physical
adsorption.
[0170] Non-specific adsorption of proteins on the slide surface is
a well-known problem in experiments associated with protein
chips.
[0171] The inventors have solved the problem by introducting
inductively coupled plasma-chemical vapor deposition (ICP-CVD).
[0172] It can be seen confirmed from the results that the ICP-CVD
is practically applicable to protein microarray chips.
[0173] The method of the present invention prevents non-specific
adsorption of proteins on the slide surface, thereby ensuring
improvement in reliability of gene diagnosis techniques.
[0174] In addition, the method enables quick and uniform deposition
of films on large-area substrates, thus realizing mass-production
and commercialization.
[0175] Although the present invention has been described herein in
detail with reference to its preferred embodiments, those skilled
in the art will appreciate that these embodiments do not serve to
limit the invention and that various changes and modifications may
be made thereto without departing from the spirit and scope of the
invention as defined in the appended claims.
* * * * *